STAT6 and STAT1 are essential antagonistic regulators of cell survival in classical Hodgkin lymphoma cell line.

Classical Hodgkin lymphoma (cHL) is a malignant lymphoid disorder characterized by aberrant activation of signaling pathways. Constitutive activation of several components of the Janus Kinase/Signal Transducer and Activator of Transcription (JAK/STAT) pathway has been observed in Hodgkin and Reed/Sternberg cells, the tumor cells of cHL. In this study, we investigate the function of STAT6 in cHL cell lines and show that STAT6 promotes survival of these cells. Microarray expression analysis of STAT6-shRNA (short hairpin RNA)-expressing cHL cell lines was carried out to analyze the STAT6-mediated survival mechanism. Some of the identified genes with potentially important regulatory functions were also interleukin (IL)-4 dependently regulated in Ramos B cells and binding of STAT6 to the regulatory regions of several genes could be confirmed, indicating that these are direct STAT6 target genes. Importantly, STAT6 knockdown increased the expression and activation of STAT1 as well as the expression of known STAT1 target genes, indicating a cross-regulation between these signaling molecules. Forced expression of STAT1 was able to induce apoptosis in cHL cell line L1236. These findings indicate that both STAT6 and STAT1 can act as important antagonistic regulators in the pathogenesis of cHL.

Transcription linked to recombination: a gene-internal promoter coincides with the recombination hot spot II of the human MLL gene.

The MLL gene is frequently involved in chromosomal translocations associated with high-risk acute leukaemia. Infant and therapy-related acute leukaemia patients display chromosomal breakpoints preferentially clustered in the telomeric portion of the MLL breakpoint cluster region (SCII). Here, we demonstrate that SCII colocalizes with a gene-internal promoter element in the mouse and human MLL gene, respectively. The mRNA generated encodes an N-terminally truncated version of MLL that still exhibits many functional regions, including the C-terminal SET-domain. Etoposide-induced DNA double-strand breaks colocalize with the binding site of RNA polymerase II and the transcription initiation region, but not with a nearby Topo II consensus sequence. Thus, the observed genomic instability of the human MLL gene is presumably linked to transcriptional processes. The consequences of this novel finding for the creation of chromosomal translocations, the biology of the MLL protein and for MLL-mediated acute leukaemia are discussed.

The role of STATs in inflammation and inflammatory diseases.

The immune response is regulated by the concerted action of pro- and anti-inflammatory cytokines. The deregulation of this process causes immunological disorders like allergic and autoimmune diseases. The Janus Kinase (JAK)--Signal transducer and activator of transcription (STAT) pathway is one major signaling pathway converting the cytokine signal into gene expression programs regulating the proliferation and differentiation of the immune cells. Several members of the STAT protein family in particular STAT1, STAT2, STAT3, STAT4 and STAT6 act as transcription factors in modulating pro- and anti-inflammatory responses. Here we review the evidence for the involvement of the different STAT proteins in inflammation, autoimmune and allergic diseases. We discuss novel approaches to interfere with the function of these signaling transcription factors for therapeutic purpose.

Transcriptional activation by STAT6 requires the direct interaction with NCoA-1.

Signal transducer and activator of transcription 6 (STAT6) is a transcription factor that is activated by interleukin-4 (IL-4)-induced tyrosine phosphorylation and mediates most of the IL-4-induced gene expression. Transcriptional activation by STAT6 requires the interaction with coactivators like p300 and the CREB-binding protein (CBP). In this study we have investigated the function of the CBP-associated members of the p160/steroid receptor coactivator family in the transcriptional activation by STAT6. We found that only one of them, NCoA-1, acts as a coactivator for STAT6 and interacts directly with the transactivation domain of STAT6. The N-terminal part of NCoA-1 interacts with the far C-terminal part of the STAT6 transactivation domain but does not interact with the other members of the STAT family. This domain of NCoA-1 has a strong inhibitory effect on STAT6-mediated transactivation when overexpressed in cells, illustrating the importance of NCoA-1 for STAT6-mediated transactivation. In addition, we showed that both coactivators CBP and NCoA-1 bind independently to specific regions within the STAT6 transactivation domain. Our results suggest that multiple contacts between NCoA-1, CBP, and STAT6 are required for transcriptional activation. These findings provide new mechanistic insights into how STAT6 can recruit coactivators required for IL-4-dependent transactivation.

Regulation of the trans-activation potential of STAT5 through its DNA-binding activity and interactions with heterologous transcription factors.

Extracellular hormones, growth factors and cytokines relay their effects on the transcription of genes through the recognition of specific receptors and intracellular signalling molecules. Signal transducers and activators of transcription (STATs) have been recognized as crucial intracellular signalling molecules. The cytokine receptor-associated Janus kinases (JAKs) convert the latent monomeric form of the STAT molecules to the activated dimeric form through tyrosine phosphorylation. The dimers bind to specific DNA response elements and are able to induce transcription. This induction requires the full-length form of the STAT molecules. Negative regulatory potential is exerted by the short form of the molecule, which lacks the trans-activation domain. This short form is activated and dimerized, but dephosphorylation is impaired. The short form of STAT occupies the DNA-binding sites in a stable fashion and acts as a strong suppressor of wild-type action. Positive enhancement of STAT5 trans-activation potential is provided by the glucocorticoid receptor. Ligand activation of this receptor causes the formation of a complex with STAT5 and deviation to the STAT5 DNA-binding site. An additional regulatory loop is provided by the reactivation of the short form of STAT5 through glucocorticoid receptor association. Conversely, classical glucocorticoid-responsive genes are negatively affected by STAT5 activation.

Interactions in the transcriptional regulation exerted by Stat5 and by members of the steroid hormone receptor family.

The pathways which connect extracellular signals with the regulation of the activity of transcription factors are being investigated in molecular detail. Extensive progress has been made in the description of the mode of action of steroid hormones and of cytokines. Steroid hormones associate intracellularly with latent receptor molecules, cause the dissociation of masking proteins, the dimerization of receptors, and their binding to specific hormone response elements in the promoters of target genes. Cytokines also activate latent transcription factors (Stats--signal transducers and activators of transcription), but act through an enzymatic mechanism. Tyrosine kinases associated with the transmembrane cytokine receptors phosphorylate Stat molecules. The phosphorylated monomers dimerize and assume specific DNA binding ability. Both classes of transcription factors bind to different response elements and regulate different target genes and both signals, cytokines and steroid hormones, can affect growth differentiation and homeostasis of different cell types. Here, we describe that Stat5, a molecule activated by several essential cytokines, functionally interacts with members of the steroid receptor family. We find that glucocorticoid receptor, mineralocorticoid receptor and progesterone receptor synergize with Stat5 in the induction of the transcription from the beta-casein gene promoter. The estrogen receptor diminishes Stat5 mediated induction and the androgen receptor has no effect. Conversely, Stat5 negatively interferes with glucocorticoid receptor, mineralocorticoid receptor and progesterone receptor induced transcription from the MMTV LTR and the estrogen receptor induced transcription from an ERE-containing promoter.

p300/CBP is required for transcriptional induction by interleukin-4 and interacts with Stat6.

Interleukin-4 (IL-4) induces tyrosine phosphorylation of the latent transcription factor Stat6, which mediates the transcriptional responses of IL-4. The transactivation domain of Stat6 has recently been mapped to the C-terminal region of Stat6. We have investigated the mechanism by which Stat6, through its transactivation domain, induces transcription. Previous studies have shown that diverse regulated transcription factors interact with coactivators such as p300 and CBP. We report that Stat6 used the interaction with p300/CBP to exert its stimulatory effects. Overexpression of p300/CBP increased IL-4-induced transcription of Stat6 activated reporter genes. The requirement of p300/CBP for Stat6-mediated transactivation is shown by coexpression of the adenovirus E1A protein. E1A repressed the IL-4-induced reporter gene activity, while mutants of E1A, which do not interact with p300/CBP, failed to block the IL-4-induced response. In addition, we found that the minimal transactivation domain of Stat6, when fused to the GAL4 DNA-binding domain, was repressed by E1A, whereas the fusion protein p300-VP16 increased the transcriptional activity. In two-hybrid protein interaction assays in mammalian cells, we mapped the interaction domain of CBP to a C-terminal region between amino acids 1850 and 2176, a region distinct from the interaction domain of CBP with Stat1, Stat2 or Stat5. Finally, we show that antibodies raised against p300 coimmunoprecipitated Stat6 and p300 from transfected COS7 cells and antibodies against Stat6 coimmunprecipitated endogenous Stat6 and CBP from Ba/F3 cells. Our data suggest that the transactivation domain of Stat6 makes contact with the basal transcription machinery by binding to p300/CBP.

p300/CREB-binding protein enhances the prolactin-mediated transcriptional induction through direct interaction with the transactivation domain of Stat5, but does not participate in the Stat5-mediated suppression of the glucocorticoid response.

Stat5 was discovered as a PRL-induced member of the Stat (signal transducer and activator of transcription) family. Its induction by many other cytokines and interleukins suggests that Stat5 plays a crucial role not only in mammary epithelial, but also in hematopoietic cells. Cell type- and promoter-specific functions of Stat5 are most likely modulated by the interaction with other transcription factors. We recently showed cross-talk between Stat5 and the glucocorticoid receptor. The activated glucocorticoid receptor forms a complex with Stat5 and enhances Stat5-mediated transcriptional induction. Conversely, Stat5 diminishes the induction of glucocorticoid-responsive genes. Here, we investigated the role of p300/CBP(CREB-binding protein), a transcriptional coactivator of several groups of transcription factors, in Stat5-mediated transactivation and in the cross-talk between Stat5 and the glucocorticoid receptor. p300/ CBP acts as a coactivator of Stat5. Its ectopic expression enhances PRL-induced Stat5-mediated transcriptional activation. Consistent with this observation, we find that the adenovirus E1A protein, which binds to p300/CBP, suppresses Stat5-induced transcriptional activation. This inhibition requires the Stat5 transactivation domain and the p300/CBP binding site of E1A. Coimmunoprecipitation and mammalian two-hybrid assays demonstrate a direct interaction between the carboxyl-terminal transactivation domain of Stat5 and p300/CBP. p300/CBP also positively interacts with the glucocorticoid receptor and enhances glucocorticoid receptor-dependent transcriptional activation of the mouse mammary tumor virus-long terminal repeat promoter. Overexpression of p300/CBP does not counteract the Stat5-mediated inhibition of glucocorticoid receptor-dependent transactivation, i.e. the repression of the glucocorticoid response by Stat5 is not a consequence of competition for limiting amounts of p300/CBP.

Physical interaction between retinoic acid receptor and the oncoprotein myb inhibits retinoic acid-dependent transactivation.

The c-myb protooncogene is predominantly expressed in hematopoietic cells and plays a vital role in hematopoiesis. Retinoic acid (RA) is able to induce differentiation of several hematopoietic cells. This differentiation is linked to decreased c-myb expression, suggesting that retinoid receptors (RAR/RXR) may down-regulate c-myb gene expression. Furthermore, recent data indicate that RAR inhibits the function of the Myb protein itself. In addition, the Myb-Ets oncogenic fusion protein has been shown to inhibit transcriptional activation by RAR and thyroid hormone receptor. Myb-Ets also antagonizes the biological response of erythrocytic progenitor cells to RA and thyroid hormone. This prompted us to investigate a possible cross talk between RAR and Myb. Here, we demonstrate that RA inhibits the expression of the endogenous Myb target gene tom-1. Conversely, Myb functions as a potent inhibitor of RA-induced biological responses. Functional analysis of Myb mutants in transfection studies revealed that the Myb DNA-binding domain (DBD) is necessary for repression whereas the transactivation domain is dispensable. Furthermore, we show that v-Myb and RAR interact in vitro and in vivo. This interaction requires the DBD of RAR. In contrast, glutathione S-transferase-pulldown assays with v-Myb mutants indicate that the DBD and the C terminus of Myb directly interact with RAR. Our results suggest that the physical interaction between Myb and RAR may play a role in the regulation of hematopoietic gene expression.

Cytokine receptor-independent, constitutively active variants of STAT5.

STAT (signal transducers and activators of transcription) proteins are dual function proteins, which participate in cytokine-mediated signal transduction events at the cell surface and transcriptional regulation in the nucleus. We have exploited insights into the activation mechanism of STAT factors to derive constitutively active variants. Chimeric genes encoding fusion proteins of STAT5 and the kinase domain of JAK2 have been derived. The functional properties of the fusion proteins have been investigated in transiently transfected COS cells or in HeLa cells stably transfected with STAT5-JAK2 gene constructs regulated by a tetracycline-sensitive promoter. The STAT5-JAK2 proteins exhibit tyrosine kinase activity and are phosphorylated on tyrosine. The molecules are activated through an intramolecular or a cross-phosphorylation reaction and exhibit constitutive, STAT5-specific DNA binding activity. The transactivation potentials of three constitutively activated STAT5-JAK2 variants comprising different transactivation domains (TADs) derived from STAT5, STAT6, and VP16 were compared. The chimeric molecule containing the STAT5 TAD had no or only a very low, the molecule with the STAT6 TAD a medium, and the molecule with the VP16 TAD a very high transactivation potential. Transcription from STAT5-responsive gene promoter regions of the beta-casein, oncostatin M, and the cytokine-inducible Src homology 2 domain-containing protein genes was observed. These chimeric STAT molecules allow the study of the function of STAT5 independent of cytokine receptors and the activation of other signal transduction pathways.

Functional antagonism between the retinoic acid receptor and the viral transactivator BZLF1 is mediated by protein-protein interactions.

The Epstein-Barr virus-encoded protein BZLF1 is a member of the basic leucine zipper (bZip) family of transcription factors. Like several other members of the bZip family, transcriptional activity of BZLF1 is modulated by retinoic acid receptors (RARs). We present evidence that the RAR alpha and BZLF1 can reciprocally repress each other's transcriptional activation by a newly discovered mechanism. Analysis of RAR alpha mutants in transfection studies reveals that the DNA binding domain is sufficient for inhibition of BZLF1 activity. Analysis of BZLF1 mutants indicates that both the coiled-coil dimerization domain and a region containing the transcriptional activation domain of BZLF1 are required for transrepression. Coimmunoprecipitation experiments demonstrate physical interactions between RAR alpha and BZLF1 in vivo. Furthermore, glutathione S-transferase-pulldown assays reveal that these protein-protein interactions are mediated by the coiled-coil dimerization domain of BZLF1 and the DNA binding domain of RAR alpha. While RAR alpha is unable to recognize BZLF1 binding sites, the RAR alpha can be tethered to the DNA by forming a heteromeric complex with BZLF1 bound to DNA. Tethering RARs via protein-protein interactions onto promoter DNA suggest a mechanism through which RARs might gain additional levels of transcriptional regulation.

Molecular cloning, sequencing and sequence analysis of the fox-2 gene of Neurospora crassa encoding the multifunctional beta-oxidation protein.

We present the molecular cloning and sequencing of genomic and cDNA clones of the fox-2 gene of Neurospora crassa, encoding the multifunctional beta-oxidation protein (MFP). The coding region of the fox-2 gene is interrupted by three introns, one of which appears to be inefficiently spliced out. The encoded protein comprises 894 amino acid residues and exhibits 45% and 47% sequence identity with the MFPs of Candida tropicalis and Saccharomyces cerevisiae, respectively. Sequence analysis identifies three regions of the fungal MFPs that are highly conserved. These regions are separated by two segments that resemble linkers between domains of other MFPs, suggesting a three-domain structure. The first and second conserved regions of each MFP are homologous to each other and to members of the short-chain alcohol dehydrogenase family. We discuss these homologies in view of recent findings that fungal MFPs contain enoyl-CoA hydratase 2 and D-3-hydroxyacyl-CoA dehydrogenase activities, converting trans-2-enoyl-CoA via D-3-hydroxyacyl-CoA to 3-ketoacyl-CoA. In contrast to its counterparts in yeasts, the Neurospora MFP does not have a C-terminal sequence resembling the SKL motif involved in protein targeting to microbodies.

Recombinant activation domains of virion protein 16 and human estrogen receptor generate transcriptional interference in vitro by distinct mechanisms.

Overexpression of transcriptional activators in transfection assays may inhibit their own activity or interfere with trans-activation by different sequence-specific transcription factors. In this study we show that this phenomenon of transcriptional interference (squelching) can be mimicked in vitro by adding recombinant activation domains to nuclear extracts. We demonstrate that the acidic activation domain of virion protein 16 interferes both with basal transcription from TATA-box promoters and promoters activated by various trans-activators in two different mammalian cell-free transcription systems. This suggests that virion protein 16 interacts with and thereby sequesters a basal transcription factor. In contrast the recombinant activation function 2 (AF-2) of human estrogen receptor does not affect basal promoter activity but inhibits TATA promoters activated by human progesterone receptor (hPR) or Sp 1 as well as the beta-globin and adenovirus major late promoter. By analyzing the effects of AF-2 on DNA binding of hPR and Sp1 we found that AF-2 inhibits the DNA binding activity of hPR, but not Sp1. Our data suggest that the recombinant AF-2 squelches Sp1 trans-activation by sequestering a common coactivator(s), whereas hPR function might be inhibited due to competition for a common cofactor stabilizing hPR dimers or through the formation of inactive heterodimers between AF-2 and hPR.